Wavelength-resolved fluorescence decay and fluorescence quantum yield of large phytochrome from oat shoots

Biochimica et Biophysica Acta, 786 (1984) 213-221

213

Elsevier BBA31869

W A V E L E N G T H - R E S O L V E D FLUORESCENCE DECAY AND F L U O R E S C E N C E Q U A N T U M YIELD O F LARGE P H Y T O C H R O M E FROM OAT S H O O T S JOACHIM WENDLER, ALFRED R. HOLZWARTH *, SILVIA E. BRASLAVSKY and KURT SCHAEFNER M a x - Planck- Institut ff~r Strahlenchemie, D - 4330 Mhlheim a.d. Ruhr (F. R. G.)

(Received August 15th, 1983) (Revised manuscript received November 29th, 1983)

Key words: Phytochrome," Fluorescence decay," Fluorescence quantum yield," (Oat shoot)

Measurements of both wavelength-resolved fluorescence decay and fluorescence quantum yield of large (119 kDa) Pr phytochrome and small (60 kDa) Pr phytochrome for the first time provide a consistent set of the photophysical parameters of the excited state of Pr" At 275 K, the lifetimes of both large and small Pr have been determined to be 45 + 10 ps, while the fluorescence yields are 2 . 0 . 1 0 - 3 and 1.5 • 10-3, respectively.

Introduction Several groups have reported fluorescence data of small and large Pr samples from various plant origins, measured under various conditions and with instruments of various sensitivities. Fluorescence spectra were qualitatively described by Correll et al. [1] for small Pr from rye in buffer solution at room temperature, and by Hendricks et al. [2] for small Pr in buffer solution and for in vivo P , both from 77 K to room temperature. The claim by Tobin and Briggs [3] that conventionally purified large P~ from rye and small P~ from oat were nonfluorescent must be attributed to insufficient instrumental sensitivity. The fluorescence data which have been reported so far are contradictory. On the one hand, Song et al. [4-8] have measured a quantum yield, ~f of approx. 0.01 for small P~ at room temperature, and ~bf~< 10 - 4 for large P~ at the same temperature, increasing to 0.030 at 200 K and reaching 0.038 at 14 K. The authors suggested that the difference in * To whom correspondence should be addressed. Abbreviations: Pr, red absorbing phytochrome; Pfr, far-red absorbing phytochrome; 0167-4838/84/$03.00 © 1984 Elsevier Science Publishers B.V.

q~f values between small and large Pr could be used to differentiate between the two forms of Pr" Hermann et al. [9], on the other hand, have recently reported ~f--(1.4 + 0.2). 10 -3 for large Pr at 293 K. Reports on fluorescence lifetime data for Pr are conflicting as well. In one report [7], the values determined for large and small P~ from rye in glycerol buffer were rf = 0.8 and 1.5 ns, respectively, at 199 K and ~'f ~< 0.2 and 0.8 ns, respectively, at 298 K. In each case, a single exponential decay was established by phase-shift measurements at two modulation frequencies (10 and 30 MHz). In another report [10], of work with three modulation frequencies (78.4, 156.8, and 235.2 MHz), average lifetimes from a non-single exponential decay were obtained for large Pr from rye in aqueous Tris-HCl buffer at room temperature. We present now a fluorescence study of large Pr phytochrome from the oat plant, including the determination of component-resolved fluorescence lifetimes and quantum yields. We also demonstrate that, in aqueous buffer solution at 275 K, the fluorescence quantum yields and lifetimes of the large (119 kDa) and small (60 kDa) phytochro-

214 mes are quite similar. It will become clear from our results that a component-resolved fluorescence decay provides a far better characterization of the state of the sample, possible impurities and, last but not least, the real photophysical parameters of Pr, than does a fluorescence spectrum alone. Materials and Methods

Phytochromes The small phytochrome (60 kDa) was extracted from etiolated oat shoots and purified as described previously [11]. The ratio A660/A280 of the samples was approx. 1.3. The large phytochrome (119 kDa), also from etiolated oat shoots, was purified on immunoaffinity columns according to Hunt and Pratt [12]. Neither ethylene glycol nor glycerol was added to the solutions. All phytochrome samples were handled under green light in 50 mM aqueous potassium phosphate buffer (pH 7.8) with 1 mM EDTA added. The sample solutions used had an absorbance of approx. 0.04-0.1 cm 1 at the absorption maximum of Pr. The phytochrome samples were fully photoreversible before and after each measurement of fluorescence quantum yield and fluorescence decay. Continuous SDS electrophoresis [13] performed on glass plates with gels of 5% (w/v) acrylamide and 0.14% (w/v) N,N'-methylenebisacrylamide, using Aldolase (158 kDa) and bovine serum albumin (68 kDa) from Boehringer Mannheim, for calibration, gave only one band, corresponding to 119 kDa. The purity index (A660/A280) was in the range 0.70 to 0.75. Interference filters (Schott) with ×max = 660 or 730 nm were inserted into the collimated beam from a 250 W iodine-tungsten lamp (Baird Atomic) for photoconversion.

Lifetime measurements Instrumentation. The fluorescence lifetimes were measured by the singe-photon timing technique. The excitation pulses were provided by a synchronously pumped mode-locked and cavity-dumped dye laser system (Spectra Physics), with an Ar + laser as the pumping source. The dye laser was operated with rhodamine 6G at a repetition rate of 400 kHz giving pulses of 5-15 ps duration (fullwidth half-maximum). The excitation wavelength

was 640 nm, and the average power was approx. 1 mW, 50% of which was used for most measurements. The detection system was a picosecond singlephoton timing apparatus capable of reliably measuring lifetimes below 20 ps [14]. The instrumentation will be described elsewhere in all detail (unpublished data). The fluorescence was selected by a double monochromator; bandwidths of 4 nm and, for the intensity studies, of 8 nm were chosen. A red-sensitive photomultiplier with a multialkali photocathode was used for detection. A width of approx. 200 ps (full-width half-maximum) was measured by single-photon timing for the excitation function. This single-photon timing arrangement combines picosecond time resolution and high sensitivity with a dynamic range of more than three orders of magnitude. These features are prerequisite for analyses of complicated decays with components of largely different amplitudes [15]. The lifetimes, analyzed in terms of a sum of exponential functions,

l(t)= L Rie r/v, i-I

were calculated by an iterative convolution procedure. The quality of the fits was judged on the basis of both a reduced X2 criterium and a plot of the weighted residuals [16]. Measuring procedure. All measurements were carried out at 275 _+ 1 K. The sample solution (3-4 ml) was contained in a thermostatically controlled reservoir, where it could be irradiated. The reservoir was connected to a thermostatically controlled measuring cuvette (cross-section 1.5 × 1.5 mm, height 10 mm). The laser beam had a crosssection of approx. 1 x 2 mm. The sample flowed, under air pressure, at a speed of 18 c m / m i n through the measuring cuvette into a second thermostatically controlled reservoir. For each lifetime measurement, one pass was carried out. The sample was then pumped back int the first reservoir where it was photoconverted back to the initial phytochrome form prior to the next measurement.

Fluorescence quantum yield measurements The fluorescence quantum yields were measured on a computer-controlled Spex Fluorolog

215 instrument [17]. For the static emission spectra, which were measured at 2 7 5 + 1 K in 1 m m cuvettes, the bandwidths were 5 nm in the excitation m o n o c h r o m a t o r and 2.5 nm in the emission m o n o c h r o m a t o r . The spectra were corrected for wavelength dependence of photomultiplier sensitivity and lamp intensity. Relative lamp intensities were measured with a rhodamine B q u a n t u m counter solution below 600 nm and with a calibrated radiometer ( E G + G model 550-1) at longer wavelengths [17]. The correction function for the emission channel was determined using a calibrated iodine-quartz lamp (1000 W, Osram). R h o d a m i n e 101, ~ f = 1.0 [18] and cresyl violet, q,r = 0.54 [19] in methanol were used as the standards for the relative q u a n t u m yield determinations. With both standards, the same ¢f values were obtained. For excitation within the ultraviolet absorption band, a degassed ethanol solution of 9,10-diphenylanthracene was taken as the standard [20]. Absorbances of sample and reference were matched at the respective excitation wavelengths. The areas of the emission spectra were integrated in the range 6 5 0 - 8 5 0 nm. N o correction was made for the refractive index difference between standard and sample solutions.

300

350

Wavelength, nm 400 500

6 0 0 700 8 0 0

I' c

o c

f //

oU (1)

:[1

xem= 6 9 5 nm

f- ~,

/ ,.,

/ %%

II

\l

x e x c = 6 4 0 nI/m

~ '~

II

~

Results and Discussion

Fluorescence yields of small and large P, Fig. 1 shows the corrected emission and excitation spectra of large phytochrome. The fluorescence q u a n t u m yields of large and small Pr at two different excitation wavelengths at 275 K are listed in Table I. The value of small Pr when excited in the first absorption band, q'r = 0.005 + 0.001, agrees reasonably with that (el --- 0.01) reported by Song et al. [7], Also, our value of large Pr, ~ = 0.0033 + 0.0003, corresponds to what could be expected from interpolation of the measured values reported by the same authors for this type of phytochrome. In fact, our values for small and large Pr at 275 K are very similar, which is at variance with a large difference reported earlier [5,7] for 300 K. The q u a n t u m yields of small and large Pr seem to depend somewhat on the absorption band within which the Pr phytochromes are excited (Table I). Thus, the overlap between the experimental errors of the ,#f values with XeXc 380 and 640 nm is only very small and the average values differ significantly. This is consistent with the independent observation that the blue-to-red ratio of the intensities is larger in the corrected fluorescence excitation spectra than in the absorption spectra. One possible reason m a y be the source of the ' a n o m a lous' emission [21]. It must contribute to the absorption of the sample at ~yxc 380 nm, and it must do so more in small than in large P~, in accord with the present measurements. It is not possible to correct for this contaminant absorption until the absorbance of the source is known. A n o t h e r reason for the observed difference may be found

TABLE I FLUORESCENCE QUANTUM YIELDS OF SMALL (S) AND LARGE (L) Pr PHYTOCHROME Potassium phosphate buffer solution (pH 7.8) at 275 K.

CV_

11

\ x ",~

30000

,J

25000 20000 -1 W a v e n u m b e r s . cm

15000

Fig. 1. Corrected fluorescence ( ) and fluorescence excitation (. . . . . . ) spectra of large P~ phytochrome in potassium phosphate buffer (pH 7.8) at 275 K.

Mxc

e~f(exptl.) ( × 10s)

chf(correc.) ( X 10 3)

(nm)

S_Pr

L_Pr

S.Pr

L-Pr

640 370

5 +1 3.4+0.7

3.3+0.3 2.9+0.3

1.5+0.3

2.0+0.2

a

a Corrected for impurity emission; cf. text and Table VI.

216

.0

I

.0

""

I

"'

2 exponentials Chi2:2.326

I

f, r

,,

_5.0 /

q' "] fr,

"'

3 exponentiols Chi~: 1.248 exponentiol: Tl(ns): T2(ns):

~'~

1.402 RI: 0.010 0.289 R 2 : 0 . 0 7 7

5

~2 6~ 1

1

~ 7

2

3

-

-

4 Time,

5

6

7

8

ns

2 exponentiols Chi2:2.540

?°/ "'"'"

•

_5.0 / 4

0

Fluorescence decays of large Pr Fig. 2 shows the semilogarithmic plots of the fluorescence decays of large phytochrome detected at 680 nm upon excitation at 640 nm. The decay curve A was obtained when the sample solution had first been subjected to saturating far-red illumination with Xirr = 730 nm. The decay observed represents therefore that of the pure Pr form. The decay curve B resulted after the sample had been converted to the photostationary equilibrium Pfr/Pr = 4 : 1 at xirr 660 nm [22]. Fluorescence decays were recorded analogously at ~m = 660, 700 and 750 nm for both the far-red- (Xirr = 730 rim) and the red- (Xirr= 660 nm) light-adapted states (Tables II and III, respectively). All the fluorescence decays measured clearly deviated from single exponential functions. They rather require a triexponential model for analysis. In the far-red light adapted state the main component with the largest relative amplitude (R 1 = 88-96%) decays with a lifetime value of 45 + 10 ps (average from several measurements). An intermediate component has a lifetime in the range of 3 0 0 + 30 ps ( R 2 = 3 9%), and the longest-lived component decays with a lifetime of 1.4 + 0.05 ns ( R 3 ~< 1 % ) (see Table II). The lifetimes of the main decay component of the red and the far-red light adapted states are identical within the error limits. The lifetime of the intermediate component of the red-light-adapted state is significantly longer, i.e., 550 + 50 ps, and the longest-lived component of this state has lifetimes in the range of 1.4 _+ 2.1 ns. These data are valid for all of the various emission wavelengths except for ?dm = 660 nm, at which wavelength the lifetimes of all decay components of both states =

0

b~ --5.0

in the presence of red-emitting compounds other than phytochrome (see below), i.e., partially or fully denatured pigment proteins.

3 e×ponentols Chi : 1.105 ,~t~

1

exponential: Tl(ns):

2

.3

4 5 Time , ns

1..396 RI: 0.042

6

7

Fig. 2. Semilogarithmic plot of the fluorescence decay of large phytochrome; AeXc = 640 nm, h em = 680 nm. (A) decay of pure Pr after saturating with far-red illumination (;?rr = 730 nm); (B) decay of an approx. 4 : 1 mixture of Pfr and Pr after saturating red illumination (Xir~= 660 nm). Note: The semilogarithmic plots of the exciting pulse (narrow band), the measured decay

(with noise), and the decay function calculated from the best-fit kinetic parameters (smooth) are shown. In the inset the calculated lifetime T 1 . . . T , and amplitudes Rj . . . R , of the decay components are given. Immediately above, a weighted residuals plot indicates the deviations of these computer-fitted parameters from the measured decay, with the X 2 value in the inset. On top, a similar plot for an n - 1 component analysis is added for the purpose of comparison.

217

TABLE II FLUORESCENCE DECAY OF T H E FAR-RED-LIGHTADAPTED FORM OF LARGE PHYTOCHROME AS A F U N C T I O N OF EMISSION WAVELENGTH Measurements taken after saturating irradiation with ?~irr= 730 nm. Potassium phosphate buffer solution (pH 7.8) at 275 K; ~cxc= 640 nm. Fluorescence lifetimes, ~'1, relative amplitudes, R1, and relative quantum yields, qh, of the Pr form; indexes 2 and 3 denote corresponding values of minor components; R; and q~i are normalized to 10070; mean lifetimes, ~'m~, are calculated from Eqn. 1. The errors of the lifetimes are 1070 or_+ 10 ps, whichever is larger. Emission wavelength, ~em: 660 nm

680 nm a

700 nm

750 nm

T1, ps R1, % '~1, %

83 88 39

48 91 54

30 95 58

43 96 62

~'2, ps

593

289

275

338

R2,%

9

8

5

3

q~2, %

30

28

26

16

03, ps R3, % q~3, %

2148 3 31

1402 1 18

923

352

~'mean, ps

1402 0.6 16 381

1451 1 22 398

a Same measurements as in Table IV at 5•0; see also Fig. 2A.

are significantly longer than at the longer emission wavelengths (Table II). The fluorescence decays do not depend significantly on the excitation intensity. This fact, amongst others, argues against the a priori possibility that the nonexponential nature of the decays could have been caused by fluorescence originating from intermediates in the photochromic transformation Pr ~ Pfr" When the intensity of the exciting light was varied over a range of 20-200% of that used for measuring the decays given in Fig. 2 and Table II and III, the results remained the same within the error limits (cf. Tables IV and V). Moreover, the flow rate of the solution in the cuvette was sufficiently high to avoid any appreciable conversion during the measurement. This is demonstrated in Fig. 3 which shows the absorption spectra of a Pr solution before and after the measurement with the normal 100% excitation intensity. The conversion was only approx. 10% of the maximum possible. Another control experiment, in which the sample solution was kept stationary in the cuvette, gave similar fluorescence results. It is possible to calculate a mean lifetime from the three decay components according to Eqn. 1. 3

y " Ri,ri 2 i=1

TABLE III Tmean F L U O R E S C E N C E D E C A Y OF T H E R E D - L I G H T ADAPTED FORM OF LARGE PHYTOCHROME AS A F U N C T I O N OF EMISSION WAVELENGTH Measurements after saturating irradiation with ~kirr= 660 nm. See text to Table II for details Emission wavelength, ~em. 660 nm

680 nm a

700 nm

750 nm

~'~, ps R 1, % qh, %

62 79 16

45 91 32

45 90 32

46 91 33

P2, ps q~2, %

789 15 42

527 5 21

557 8 33

580 6 28

r a, ps

1 967

1 395

2073

1 517

R3,%

6

4

2

3

q~3, %

42

47

36

39

1 151

760

890

746

R 2, %

~'mean, ps

Same measurements as in Table V at 101o; see also Fig. 2B.

3

(1)

i=l

These mean lifetimes are given in Table II and III for the various emission wavelengths. They should be compared with the single-lifetime data obtained by Hermann et al. [10] with the phase shift technique. These and our calculated mean lifetimes show a roughly parallel dependence on the emission wavelength. However, it has now become clear that a single mean lifetime has no meaning as a real photophysical property of phytochrome. Only the component of approx. 45 ps (average value) should be attributed to the excited state of Pr for the following reasons. Firstly, it comprises more than 90% of the relative decays amplitude. Taking into account the strong overlap of the emission spectra of the various components (cf. Table II0 and assuming similar extinction coeffi-

218 T A B L E IV FLUORESCENCE DECAY OF THE FAR-RED-LIGHTA D A P T E D F O R M O F L A R G E P H Y T O C H R O M E AS A FUNCTION OF EXCITATION INTENSITY

0.08

Measurements after saturating irradiation with ~irr = 730 nm; h em = 680 rim. 51 o is the standard intensity used for the meas u r e m e n t of the emission wavelength dependence in Table II. See text to Table II for details

0.06 Excitation intensity 10

510 ~

101 o

R1, % ~1, %

62 96 72

62 96 69

48 91 54

41 96 70

~ , pS R 2, % ~2, %

364 4 17

370 17

289 8 28

330 3 15

~ , ps R 3, % ~3, %

2148 0.1 11

1732 0.1 14

1 402 1 18

1494 1 15

rl, ps

21 o

4

" Same measurements as in Table II with ; ~ m = 680 nm; see also Fig. 2A.

cients, the relative amplitude of a decay component in any experiment may be taken as an approximate measure of the relative ground-state

TABLE V FLUORESCENCE DECAY OF THE RED-LIGHTA D A P T E D F O R M O F L A R G E P H Y T O C H R O M E AS A FUNCTION OF EXCITATION INTENSITY

Measurements after saturating irradiation with xirr= 660 nm; ~cm= 680 rim. 1010 is the standard irradiation intensity used for the measurements of the emission wavelength dependence in Table 111. Excitation intensity 10

210

1010 a

T1, ps R I, % if1, %

67 92 48

60 89 38

45 91 32

~ , ps R 2, % ~2, %

440 5 18

418 7 20

527 5 21

1522 3 34

1494 4 42

1395 4 47

~,ps R3,% ~3, %

Same measurements as in Table III with X~ m = 680 nm; see also Fig. 2B.

u c o JD L_

o .£)

0.04 <

0.02

I 500

I 600 Wavelength,

700

800

nm

Fig. 3. Absorption spectra of large phytochrome, taken after fluorescence lifetime measurement. (a) Absorption directly after the decay measurement, starting from the pure Pr form. (b) Absorption after saturating irradiation with ~kirr = 730 nm.

concentration of the corresponding species. This means that the 45 ps lifetime belong to the main ground-state species. The assignment of the shortest-liver decay component (~-~ = 45 ps) to the singlet decay of excited Pr is further supported by the results of fluorescence decay measurements on the same phytochrome sample after a saturating red light irradiation (~irr = 660 rim) (cf. Table III). It is this decay component whose lifetime remains constant upon red/far-red irradiation. The relative amplitude of the longest-lived decay component was much stronger in the Pfr state than in the case with pure Pr- Since the absolute yield of the longest-lived decay component seems to be invariant with the irradiation conditions (i.e., the PJPfr ratio), we may take this value as an internal standard for the comparison of the integrated intensities of the short-lived decay components. The ratio of relative yields of the shortest-lived (45 ps) decay component in the far-red- to red-lightadapted state has a value of approx. 4, independent of the emission wavelength. Within the ex-

219 perimental error, this ratio is in agreement with the theoretical ratio of 4 expected from the known 1 : 4 composition of P~ and Pf~ at photoequilibrium with h, rr= 660 n m [22]. The intermediate component does not behave accordingly. The two longer-lived c o m p o n e n t s must therefore be considered to be 'impurities' of some kind. These ' i m p u r ities' which give rise to a non-single exponential fluorescence decay seem to be characteristic of such p h y t o c h r o m e preparations, as is suggested by similar observations of H e r m a n n et al. [9,10]. It is believed that these 'impurities' stem from partially denatured p h y t o c h r o m e molecules that still contain the tetrapyrrole chromophore. The c o m p o u n d that gives rise to the middle decay c o m p o n e n t (~2, cf. Tables II and III) even retains photoreversibility. This is revealed by the reversible appearance of decay c o m p o n e n t s with lifetimes of ~'2 of approx. 300 and approx. 550 ps upon far-red or red irradiation, respectively. It is i m p o r t a n t to note that our method of measuring the component-resolved fluorescence decays and the relative amplitude of the c o m p o nents directly provides a sensitive measure for the relative a m o u n t of these 'impurities'. The data given in Table II reveal that the purity of our preparation, as judged from the R~ value, is about 90%. Most other test methods used conventionally would p r o b a b l y fail to detect impurity levels of a few percent. Despite this relatively high purity,

a b o u t 40% of the total integrated fluorescence of large (119 kDalton) Pr is due to impurities because of their much longer lifetimes as c o m p a r e d to the Pr lifetime. The experimentally determined fluorescence yield q~f(exptl) from an ordinary C W spectrum (Table I) comprises the contributions of all emitters present in the solution. To arrive at the real fluorescence yield of Pr we have to subtract the relative yields of the impurities as determined from the time-resolved experiments (Table II). We thus arrive at a fluorescence yield for Pr corrected for impurities of ,#f(corr)= (2 + 0.2). 10 -3 (Table VI). A radiative lifetime for the S 1 state of Pr of ~'raa = 20 ns can be derived from the Pr oscillator strength given by Song et al. [7]. Taking our fluorescence lifetime of ~'f = 45 ps, a theoretical fluorescence q u a n t u m yield of q~f(calcd)= (2.3 + 0.4). 10 -3 can be calculated. This value is in excellent agreement with our impurity-corrected fluorescence yield, ,~f(corr)= (2.0 _+ 0.2). 10 -3, which was obtained from our independently measured q,r(exptl) = (3.3 _+ 0.3). 10 -3 (Table I). The set of photophysical parameters for the S 1 state of P~ can thus be c o m p l e m e n t e d as summarized in Table VI. C o m b i n a t i o n s of measured q~f and ~'f values from other authors [5,7,9,10] according to the equation ~f = k~aa.~'r, where k~aa is the radiative lifetime, generally give k~, d values differing in up to three orders of magnitude from the known [7] 20 ns

TABLE VI PHOTOPHYSICAL PARAMETERS OF THE

S1

STATE OF LARGE Pr PHYTOCHROME AT 275 K

Parameter Radiative lifetime Fluorescence lifetime Fluorescence quantum yield (Xe×c= 640 nm) uncorrected for 'impurity emission' a corrected for 'impurity emission' b Calculated fluorescence quantum yield Quantum yield of the Pr -~ Prr phototransformation a cf. Table I. b cf. text.

Source Trad = 20 ns ~-f= 45 _+10 ps

[9] this work

~f = (3.3 _+0.3)-10- 3

this work

t~f = (2.0 __+0.2)' 10- 3

this work

~f = (2.3 4-0.4). 10- 3

this work

q~eac= 0.17 q,.... = 0.11

[23] [25]

220 value. Our data are gratifying in the sense that they provide the first consistent set of photophysical parameters of the excited state of Pr. The sum of the overall photochemical yield of the Pr---, Prr conversion (~pc = 0.11 0.17) [23,24] and the fluorescence yield of Pr is considerably smaller than unity. Two possibilities may be considered for this behaviour. Either a fast direct internal conversion occurs from the Pr excited state to the ground state with a maximum ~,c of 0.8, or the primary photochemical step forming lumi-R, the first intermediate in the Pr ---' Pfr photoconversion, occurs with almost unit quantum yield whereupon partial dark reversion would occur from one of the intermediates. We prefer the first alternative, since no indications for thermal reversion have been found in flash photolytic experiments. Our %...... values as a function of emission wavelength (Table II) agree fairly well with the phase shift-measured lifetimes reported by Hermann et al. While their lifetime changes from approx. 600 ps to 300 ps with increasing detection wavelength from 650 to 710 nm, our ~m.... value is almost constant in the range )~enl= 680--750 nm and merely increases to approx. 900 ps at X~m= 660 nm. The differences can easily be explained by the fact that in the phase shift technique the effective lifetime is dependent also on the modulation frequency [10]. It has already been pointed out by the authors [10] that their phase-shift-measured lifetime is only an average value which may be ascribed to two or more exponential decay processes.

Fluorescence decay of small Pr Fluorescence decay measurements have been carried out on red- and far-red-light-adapted samples of small P~ (60 kDa) in the same way as described above for large P~. No intensity dependence measurements have been performed, however. It was found that small Pr had a decay characteristic similar to that of large Pr. Again we find a contribution of long-liver impurities to the fluorescence decay, which does not follow a single exponential function. It is important, however, to note that the lifetime of the short decay component, i.e., the one attributable to the chromophore of small Pr (~'~ = 45 _+ 10 ps) is identical to that of large P~. From the relative contribution of the long-lived components to the total fluorescence

yield it is now also possible to calculate a corrected yield of ~ f = 1.5.10 ~ (cf. Table I) for small Pr. Thus small and large P~ have identical photophysical parameters within the error limits. Conclusion It has been shown that the conflicting values of both fluorescence yields and lifetimes reported for phytochrome in the past have been due to the contribution of unresolved fluorescence components with largely longer lifetime and thus higher quantum yield that those of Pr itself. This problem could be solved only by wavelength and decay component-resolved measurements of fluorescence lifetimes. Pfr most probably has a lifetime considerably shorter than 20 ps and no fluorescence has been observed from this form. It is now known that the large degraded '119 kDa' Pr in fact consists of a mixture of 114 and 118 kDa phytochromes [25]. On the basis of our fluorescence decay measurements, however, we believe this phytochrome to be essentially homogeneous as far as the photophysical parameters are concerned. This is not in disagreement with the somewhat longer decay time (up to 60 ps) found at the short-wavelength edge of the fluorescence spectrum ()~ ..... = 660 nm). Such type of 'heterogeneity' is not unexpected with molecules of this flexibility and it is still consistent with the fully functional state of both chromophore and apoprotein. Our interpretation is also supported by the fact that the small (60 kDa) Pr has identical photophysical parameters. The considerably longer decay times (above 250 ps) must clearly be ascribed to components denatured to various extents. The fluorescence yield which is in best agreement with our impurity-corrected value is that, albeit from rye Pr, reported recently by Hermann et al. [9] at 293 K. The relatively minor differences between these two measurements can be attributed to the slightly different measuring conditions, especially the different temperature. It is now widely accepted that the 124 kDa phytochrome represents the native form of phytochrome [23,25]. This phytochrome form has different photochemical properties. It will be interesting

221 to e x t e n d m e a s u r e m e n t s o f the k i n d r e p o r t e d h e r e a l s o o n s a m p l e s o f such n a t i v e Pr" Acknowledgements T h e s a m p l e s o f small a n d large p h y t o c h r o m e u s e d in this s t u d y h a v e b e e n isolated, as p a r t of a c o l l a b o r a t i v e p r o j e c t , by the g r o u p of P r o f e s s o r E. Sch~fer, U n i v e r s i t y of F r e i b u r g . W e t h a n k M i s s A. Keil, M i s s D. K r e f t a n d Mr. H.V. S e e l i n g ( M i a l h e i m ) for a b l e t e c h n i c a l assistance.

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